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Biochemistry. 1993 Apr 6;32(13):3368-76.

Thioltransferase is a specific glutathionyl mixed disulfide oxidoreductase.

Author information

1
Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4965.

Abstract

To study the substrate specificity and mechanism of thioltransferase (TTase) catalysis, we have used 14C- and 35S-radiolabeled mixed disulfides of cysteine and glutathione (GSH) with various cysteine-containing proteins. These protein mixed disulfide substrates were incubated with glutathione, glutathione disulfide (GSSG) reductase, and NADPH in the presence or absence of thioltransferase. Glutathione-dependent reduction of protein mixed disulfides was monitored both by release of trichloroacetic acid soluble radiolabel and by formation of GSSG in an NADPH-linked spectrophotometric assay. GSH-dependent dethiolation of [35S]glutathione-papain mixed disulfide (papain-SSG) and the corresponding bovine serum albumin mixed disulfide (BSA-SSG) were catalyzed by thioltransferase (from human red blood cells) as shown by the radiolabel assay, and equivalent rates were measured by the spectrophotometric assay. Dethiolation of [35S]hemoglobin-glutathione mixed disulfide (Hb-SSG) was also catalyzed by TTase. In contrast, TTase did not catalyze GSH-dependent dethiolation of [14C]papain-SScysteine or [14C]BSA-SScysteine as measured by the radiolabel assay. [14C]Hb-SScysteine and Hb-SScysteamine also did not serve as substrates. In separate experiments, TTase from rat liver displayed analogous selectivity. Thus, thioltransferase (glutaredoxin) appears to be specific for glutathione-containing mixed disulfides. Apparent TTase catalysis of GSSG formation from the papain- and BSA-SScysteine mixed disulfides was observed by the spectrophotometric assay, but a lag phase occurred consistent with preenzymatic formation of GSScysteine which could serve as the actual TTase substrate. Two-substrate kinetic studies of TTase with GSH and GSScysteine gave patterns of parallel lines on double-reciprocal plots (1/V vs 1/[S]), consistent with a simple ping-pong mechanism involving a TTase-SSG intermediate.

PMID:
8461300
DOI:
10.1021/bi00064a021
[Indexed for MEDLINE]

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